1. Field of the Invention.
This invention discloses human tissue plasminogen activator (TPA) analogs. The analogs are TPA-like molecules that have had the native domain regions either rearranged, deleted, added or a combination thereof. The analogs are the product of the expression of recombinant deoxyribonucleic acid (DNA) also described herein. Additionally, the present invention describes replication and expression plasmids containing the TPA-coding DNA sequences described above and suitable host microorganisms that are capable of expressing the TPA analogs after becoming transformed with an appropriate plasmid.
2. Background
TPA has therapeutic value in treating blood clots by helping dissolve the clots as they form. Thrombolysis is the process of fibrin clot dissolution. For that purpose the serine protease plasmin is formed from its inactive zymogen, plasminogen. Activation of plasminogen is achieved through proteolytic cleavage by a class of serine proteases called plasminogen activators. These activators are present in most body fluids and initiate the activation of plasminogen to plasmin. This sets up a cascade whereby a small amount of plasminogen activator can initiate the activation of a large amount of plasminogen. In vivo, the plasminogen activator activates plasminogen to plasmin which then acts to degrade the fibrin clot. The plasminogen activator which is physiologically important to clot lysis is TPA.
TPA is a serine protease with a molecular weight of approximately 66,000 daltons which is produced by the vascular endothelial cells. It is glycosylated and its only known protein substrate is plasminogen. TPA has five domains: the fibronectin finger domain (F), the growth factor domain (G), the kringle 1 domain (K1), the kringle 2 domain (K2) and the active site domain (A). One or more of these domains is responsible for the unique fibrin binding activity of TPA.
In addition to its fibrin affinity, TPA activation of plasminogen is enhanced in the presence of fibrin. These properties make TPA a valuable therapeutic agent because other clot dissolving drugs such as urokinase or streptokinase do not have any specificity for the fibrin clot.
By manipulating the domains it is possible to improve the affinity of the native enzyme for the fibrin and increase its in vivo half-life. More specifically, duplication of the Kringle 2 or Finger domain will enhance fibrin affinity. Elimination of the growth factor domain will enhance in vivo half-life and places the finger region domain next to the kringle regions 1 or 2 to increase fibrin affinity. Information Disclosure.
Human TPA has been purified and its physical characteristics have been studied. Rijken et al. "Purification and Characterization of the Plasminogen Activator Secreted by Human Melanoma Cells in Culture", J. Biol. Chem., vol. 256, no. 13, pp. 7035-41 (July 10, 1981). Experimental quantities of human TPA are obtainable from the culture medium of human melanoma cells. Kluft, C. et al., "Large-scale production of extrinsic (tissue-type) plasminogen activator from human melanoma cells" in Advances in Biotechnological Processes, Eds. Mizrahi, A. and Wezel, A. L. 2:97-110 (1983). The bioactivity of human TPA has been studied, and it has been demonstrated to have therapeutic value in animal models and in humans through its ability to dissolve life-threatening blood clots. Korninger et al., "Thrombolysis with Human Extrinsic (Tissue-type) Plasminogen Activator in Dogs with Femoral Vein Thrombosis", J. Clin. Invest., vol. 69, pp. 573-580 (March, 1982); Weimar et al., "Specific Lysis of an Iliofemoral Thrombus by Administration of Extrinsic (Tissue-type) Plasminogen Activator", The Lancet, pp. 1018.20 (Nov. 7, 1981); and Van De Werf, F. et al., Coronary Thrombolysis with Tissue-type Plasminogen Activator in Patients with Evolving Myocardial Infarction, J. of N. Eng. Med., 310:609-613 (1984). The half-life of TPA is estimated at 2-3 minutes making it inconvenient, if not impractical, to use as a therapeutic agent. Collen, D. et al., Clot-selective Coronary Thrombolysis with Tissue-type Plasminogen Activator, Science, 220: 1181-1183 (1983).
The enzyme kinetics of TPA have been studied. Rijken et al., "Fibrinolytic Properties of One-Chain and Two-Chain Human Extrinsic (Tissue-type) Plasminogen Activator", J. Biol. Chem., vol. 257:2920-2925 (1982). The binding and fibrolytic affinity of human TPA for human blood clots over the blood clots of other animals is known. Korninger et al., "Studies on the Specific Fibrinolytic Effect of Human Extrinsic (Tissue-type) Plasminogen Activator in Human Blood and in Various Animal Species in Vitro", Thrombos. Haemostas., 46(2): 561-565 (1981). The structural relationship of domains with the fibrinolytic activity and affinity has also been delineated. Banyai, L. et al., Common evolutionary origin of the fibrin-binding structures of fibronectin and tissue-type plasminogen activator, FEBS Lett. 163(1):37-41 (1983) and Ny, T. et al,. The Structure of the Human Tissue-type Plasminogen Activator Gene: Correlation of Intron and Exon Structures to Functional and Structural Domains, Proc. Natl. Acad. Sci. USA 81:5355-5359 (1984).
The expression of TPA by transformed bacteria and yeast is also known. The messenger ribonucleic acid (RNA) of human TPA was first isolated in 1982. Opdenakker et al., "Messenger RNA for Human Tissue Plasminogen Activator", Eur. J. Biol., pp. 269-74 (1982). Bacteria were transformed for expression of TPA by Pennica et al., "Cloning and Expression of Human Tissue-type Plasminogen Activator cDNA in E. coli.", Nature, 301:214-21 (1983). Procedures to transform yeast to express human TPA have been described in U.S. patent application Ser. No. 663,025 and published European patent application EP 143,081.
There have been several recent reports of domain deletions of TPA in the literature using recombinant technology or other enzymatic techniques. A discussion of these reports is offered but the inventors do not concede that these references are prior art under 35 USC 102 at this time and provide them in order to offer a complete background of this invention. The first studies take advantage of native restriction sites to delete sections of the TPA protein. J. H. Verheigen, et al., Involvement of finger domain and kringle of tissue-type plasminogen activator in fibrin binding and stimulation of activity by fibrin, EMBO Journal Vol. 5 No. 13:3525-3530 (1986); A. J. van Zonneveld, et al., On the Interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin, J. Biol. Chem. Vol. 261 No. 30:14214-14218 (1986) and A. J. van Zonneveld, et al., Autonomous functions of structural domains on human tissue-type plasminogen activator, Proc. Natl. Acad. Sci. USA 83: 670-4674 (1986). There are several abstracts indicating mutants of TPA but they do not offer sufficient methodology to discern whether they have indeed disclosed deletions of the nature described herein. N. U. Bangs, et al., Functional properties of tissue plasminogen activitor mutants, Blood 66(5) Suppl. 1 Asbstract 1205 page 330a (1985); N. U. Bangs, et al., Tissue plasminogen structure-function relationships, Clin. Res. Vol.33 No.4 878A, (1985) and D. Tiemeier, et al., Structure:function studies of tissue-type plasminogen activator, Fed. Proc. 45 (4) Abstr. 4702 page 963 (1986). Others have made deletions through enzymatic degradation processes. EP 196920-A Degraded species of tissue-type plasminogen activator--useful in treating thrombotic disease and having a reduced clearance rate in vivo.